Current research investigating chimeric antigen receptor (CAR)-T cell therapeutics indicates that CAR properties other than intracellular signaling systems, such as high association rate constants and enhanced self-interaction of the antigen-binding domain (ABD), elicit enhanced therapeutic efficacy and are key factors for CAR-T candidates. However, rational strategies to achieve these features are to be established. To develop novel CAR-T cells with these characteristics, ABD was engineered by introducing several arginine residues into the light-chain framework region-3 of the single-chain fragment variable. The mutant CAR-T cells exhibited a higher cell-killing efficacy than that of wild-type cells and superior antitumor effects in mice, prolonged persistence in vivo, and decreased interferon-γ secretion. RNA sequencing revealed differential gene expression profiles between the mutant and wild-type CAR-T cells before and after antigen stimulation. In conclusion, we proposed a design strategy to generate CAR-T cells with high therapeutic efficacy by modulating CAR properties using an arginine cluster.

Background Recent progress of engineered T cells genetically modified to express tumor-specific T-cell receptors (TCR-T) has demonstrated promising perspectives for treatment of a variety of cancers. Nonetheless, the efficacy of this strategy against solid tumors is still unsatisfactory due to poor expansion, loss of function, and short persistence of the infused cells in vivo. To overcome this, different combination therapies with other modalities, including cancer vaccines and immune-checkpoint inhibitors (ICIs), have attracted attention and are currently being developed to achieve long-term patients' survival. However, the optimal strategy has not been determined yet. Recently, we newly developed a novel cancer vaccine based on hyaluronic acid nanogel (HANG-Vax). HANG can easily embed various substances, such as long peptides, leading to form nanoparticles of about 100 nano meter diameter. HANG-Vax can efficiently deliver vaccine antigens to antigen presenting cells present in lymph nodes after subcutaneous injection. Aims In this study, we investigated the efficacy of the combined treatment of HANG-Vax with TCR-T therapy by using tumor-bearing mouse models. Methods The efficacy of the combination of TCR-T and HANG-Vax therapy was examined by using each tumor bearing mouse treatment model. Here, we utilized C57BL/6-derived B16F10 melanoma cell line, which is resistant to ICIs and TCR-T therapies. TCR-T cells were prepared from splenic CD8+ T cells of Pmel-1 mice transfected with TCR that specifically recognize gp100. HANG-Vax was prepared by mixing HANG with long peptide encoding gp100. HANG-Vax was subcutaneously injected on day 7, 11 and 15, and CD8+ T cells were intravenously administered on day 8 and 12 after tumor inoculation. Along with monitoring tumor growth, single-cell RNA/TCR seq analysis and spatial gene expression analysis were performed to analyze the dynamics of the infused TCR-T cells in lymph nodes and tumor tissues. Results Malignant cell transplantation revealed that complete eradication of B16F10 tumors was observed in all mice treated with TCR-T and HANG-Vax therapy. In contrast, the other groups of mice were all dead until 25 days after transplantation of B16F10. Mice that achieved complete remission (CR) survived for long periods and exhibited vitiligo hair. Single-cell RNA/TCR seq analysis of the lymph nodes from each group revealed that HANG-Vax induced the clonal expansion of TCR-T, but much less in TCR-T monotherapy. Projection of reference data identified that HANG-Vax differentiated the TCR-T toward effector memory T cells. In addition, the differential gene expression analysis across the groups showed that HANG-Vax treatment induced the expression of cytotoxic genes such as Granzyme B. KEGG-pathway analysis using the differentially expressed genes highlighted the HANG-Vax accelerated not only “cell-killing”-related gene expression but also “cell metabolism/proliferation”-related genes. These suggested the clonal survival and expansion was induced in HANG-Vax. To confirm the efficient recruitment of cytotoxic T cells at the tumor site, further analysis utilizing the spatial expression analysis was performed. CD8+T cells were highly co-exisiting with Pmel expressing tumor cells in HANG-Vax treatment group. Pseudo-bulk analysis of these spatial expression data showed significantly higher levels of “cytotoxicity” and “positive activation of immune cell”-related genes. Regarding the non-T population of tumor site, we performed deconvolutional analysis of each spot using Deepcolor method. Interestingly, TCR-T with HANG-Vax induced the higher expression of chemokines, including Ccl4, Ccl5, Cxcl9, and identified that HANG-Vax treatment group possessed higher macrophage invasion with M1 macrophage, suggesting HANG system empowered the macrophage-related anti-neoplastic effect in addition to CD8+ T cell cytotoxicity. Summary/Conclusion These results indicate that HANG-Vax can potently maximize the efficacy of TCR-T therapy, leading to the cure of immunotherapy-resistant solid tumors and long-term prevention of tumor recurrence. Besides, HANG-Vax can potentially transform the characteristics of resistant-tumors, rendering them more sensitive to immunotherapy. We believe that this study may provide important insights into the clinical application of HANG-Vax combined with adoptive cell therapy for patients with solid tumors.

Abstract Background: Cholesterol-conjugated hyaluronic acid, synthesized by grafting cholesterol moieties onto hyaluronic acid, spontaneously formed nano-sized hydrogel of approximately 30-100nm in water. This hyaluronic acid derivative (HA nanogel) has demonstrated the ability to encapsulate various modalities, including low- and medium-sized molecules, peptides, and proteins. Previous reports have highlighted its efficacy in solubilizing poorly water-soluble drugs and serving as a sustained-release matrix. In this study, we explore the potential of HA nanogels encapsulating antigen peptides with adjuvant as a cancer vaccine and a priming tool for combinational immunotherapy. Method: We prepared HA nanogels as a novel peptide carrier for cancer vaccines and attempted to encapsulate various peptides, including neoantigen (mERK2) peptides expressed in immune checkpoint inhibitor (ICI) therapy-resistant fibrosarcoma cell line CMS5a, gp-100 peptides expressed in metastatic melanoma cell line B16F10, and MAGEA4. Our experiments covered APC uptake, antigen-specific CTL induction, and anti-tumor studies to demonstrate the utility of HA nanogels in these applications. Results: HA nanogel efficiently formed stable complexes with mERK2, gp-100 and MAGEA4 long peptides, each with diameters of 30-100nm. The facile and simple vaccine formulation processes with HA nanogel achieved high drug content and high encapsulation efficiency of 90% or higher. Fluorescently labeled mERK2 long peptide encapsulated in HA nanogel vaccines were subcutaneously administered to BALB/c mice, and uptake analysis of mERK2 long peptides in draining lymph nodes (dLN) was performed using flow cytometry. In comparison to the group receiving mERK2 long peptide without nanogel encapsulation, the HA nanogel group exhibited higher uptake by macrophages and dendritic cells in the dLN. Administering HA nanogel vaccines containing mERK2 long peptides with CpG oligoDNA resulted in higher antigen-specific CD8+ T cells compared to the group receiving mERK2 long peptides without encapsulation. Strong anti-tumor effects were demonstrated in mice models with subcutaneously transplanted CMS5a and B16F10 cell lines. The enhanced efficacy through co-administration with ICI was also confirmed. Conclusion: HA nanogel vaccines efficiently deliver antigen peptides to macrophages and dendritic cells, inducing antigen-specific CTL responses, thereby demonstrating anti-tumor effects against cold tumors such as CMS5a and B16F10. HA nanogels have the potential not only as carriers for cancer vaccines but also as priming tools for combinational immunotherapy. Citation Format: Takashi Nakai, Fumiyasu Momose, Kohei Yabuuchi, Makiko Yamane, Tae Hayashi, Linan Wang, Yoshiyuki Nakagawa, Shogo Aso, Toru Katsumata, Tsuyoshi Shimoboji, Yoshihiro Miyahara. Crafting hyaluronic acid-based nanoparticles for enhanced LN targeting as a potent priming tool in immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6745.

Despite the revolutionary success of chimeric antigen receptor (CAR)-T therapy for hematological malignancies, successful CAR-T therapies for solid tumors remain limited. One major obstacle is the scarcity of tumor-specific cell-surface molecules. One potential solution to overcome this barrier is to utilize antibodies that recognize peptide/major histocompatibility complex (MHCs) in a T cell receptor (TCR)-like fashion, allowing CAR-T cells to recognize intracellular tumor antigens. This study reports a highly specific single-chain variable fragment (scFv) antibody against the MAGE-A4p230-239/human leukocyte antigen (HLA)-A∗02:01 complex (MAGE-A4 pMHC), screened from a human scFv phage display library. Indeed, retroviral vectors encoding CAR, utilizing this scFv antibody as a recognition component, efficiently recognized and lysed MAGA-A4+ tumor cells in an HLA-A∗02:01-restricted manner. Additionally, the adoptive transfer of T cells modified by the CAR-containing glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related receptor (GITR) intracellular domain (ICD), but not CD28 or 4-1BB ICD, significantly suppressed the growth of MAGE-A4+ HLA-A∗02:01+ tumors in an immunocompromised mouse model. Of note, a comprehensive analysis revealed that a broad range of amino acid sequences of the MAGE-A4p230-239 peptide were critical for the recognition of MAGE-A4 pMHC by these CAR-T cells, and no cross-reactivity to analogous peptides was observed. Thus, MAGE-A4-targeted CAR-T therapy using this scFv antibody may be a promising and safe treatment for solid tumors.

Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated impressive success in the treatment of patients with hematologic tumors yet achieved very limited efficacy for solid tumors due to hurdles unique to solid tumors. It is also noted that the tumor microenvironment composition varies between tumor type, which again imposes unique set of hurdles in each solid tumor. Therefore, elucidation of individual hurdles is key to achieving successful CAR-T therapy for solid tumors. In the present study, we employed an orthotopic human PDAC xenograft model, in which quantitative, spatial and functional dynamics of CAR-T cells in tumor tissues were analyzed to obtain insights into ways of overcoming PDAC related hurdles. Contrary to previous studies that demonstrated a limited persistency and infiltration of CAR-T cells in many solid tumors, they persist and accumulated in PDAC tumor tissues. Ex vivo analysis revealed that CAR-T cells that had been recovered at different time points from mice bearing an orthotopic PDAC tumor exhibited a gradual loss of tumor reactivity. This loss of tumor reactivity of CAR-T cells was associated with the increased expression of AMP-activated protein kinase and Mitofusin 1/ Dynamin-related protein 1 ratio.

Abstract Background: We have developed nanosized hydrogel particles (nanogels) to create new nanomaterials for biomedical applications. In particular, Hyaluronic Acid, partially hydrophobized by chemical modification with cholesteryl groups, has a distinguished characteristic to form physically cross-linked NanoGel particles. HANG efficiently forms a stable complex with an antigenic polypeptide (HANG-Vaccine) through hydrophobic interactions. In this study, we investigated the efficacy of HANG-V against tumors resistant to immune checkpoint inhibitors (ICIs) and adoptive TCR-T therapy. Methods: C57BL/6-derived B16F10 melanoma and BALB/c-derived CMS5a sarcoma are both known to be resistant to ICIs and adoptive TCR-T therapy. The efficacy of HANG-V was tested by using each tumor-bearing mice treatment model. HANG-V was prepared by mixing gp100 or CMS5a neoantigen (Ag) with HANG, respectively. Results: HANG-V was subcutaneously injected into B16F10-bearing C57BL/6 on days 7, 11, and 15, and CD8+ T cells from Pmel-1 mice were intravenously administered on days 8 and 12. Surprisingly, complete regression of established B16F10 tumors with more than 100 mm2 were observed. Vaccinated mice survived with changing hair color of mice into white, while non-vaccinated mice were all dead by day 25. Moreover, gp100-specific CTLs was observed abundantly in systemic including tumor site. Even two months after the treatment, transferred T cells with memory phenotype were persistently detected in peripheral bloods. Furthermore, booster vaccination elicited robust expansion of these memory T cells persisting for more than one year. In similar experimental settings, we treated CMS5a tumor-bearing mice by HANG-V combined with adoptive transfer of CD8+ T cells from DUC18 mice, whose T cells are specific for mutated ERK2 neoAg. All mice survived as well with complete reduction of established CMS5a tumors, rejected a rechallenge with CMS5a and induced an Ag-specific CTL responses on more than 2 years. Notably, by using neoantigen-knockout CMS5a, we obtained results suggesting that HANG-V with TCR-T therapy potently induced the so-called “antigen spreading”. Furthermore, induction of Ag-specific CTLs was associated with CD44 hyaluronic acid receptor which expressed in APCs crucial for cross-presentation to CD8+ T cells. Conclusions: HANG-V strongly enhanced the efficacy of adoptive TCR-T cell therapy against ICI refractory tumors leading to total tumor suppression. Furthermore, HANG-V induced potent and persistent Ag-specific CTLs systemically through interaction with CD44 hyaluronic receptor, allowing long-term protection of tumor recurrence with memory CD8+ T cells. Our studies may propose crucial insights for clinical application of HANG vaccine with adoptive T cell therapy in patient with ICI-resistant tumors with poor prognosis. Citation Format: Fumiyasu Momose, Takashi Nakai, Kohei Yabuuchi, Makiko Yamane, Tae Hayashi, Linan Wang, Yoshiyuki Nakagawa, Shogo Aso, Toru Katsumata, Tsuyoshi Shimoboji, Yoshihiro Miyahara. A novel cancer vaccine based on hyaluronic acid nanogel combined with adoptive T cell therapy induces complete regression of established tumors and long-lasting memory CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4091.

Recent studies confirm the critical roles of endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) in malignant behavior of various cancers. Nevertheless, what function ERO1L plays in lung adenocarcinoma (LUAD) remains uncovered. The expressions and clinical significance of ERO1L in LUAD were investigated using the TCGA dataset. The ERO1L levels were examined by RT-qPCR. The LUAD cell proliferation was valued using colony formation as well as CCK-8 assays. The invasion and migration abilities of LUAD cells were detected through Transwell in addition to wound healing assays. The effects of ERO1L on LUAD cell apoptosis were determined by flow cytometric analysis. Moreover, we also established mouse xenograft models of LUAD cells to confirm the functions of ERO1L in vivo. The ERO1L levels in tumors were identified by immunohistochemistry. Western blot was used for the detection of the levels of Wnt/βcatenin signaling-related proteins. The TCGA database revealed that ERO1L expressions were higher in LUAD tissues than those in non-cancerous tissues. ERO1L overexpression was related to poorer overall survival of LUAD patients. In addition, ERO1L silence suppresses LUAD cell clone formation, proliferation, migration as well as invasion but induces apoptosis. Moreover, we also verified that ERO1L silence could promote LUAD growth in vivo. Based on the mechanism analysis, ERO1L was confirmed to regulate LUAD development via Wnt/βcatenin cascade signal. ERO1L, the expression of which was increased in LUAD tissues, functioned as an oncogene. ERO1L silence significantly attenuated LUAD tumorigenesis, likely via inhibition of Wnt/βcatenin signaling, indicating that ERO1L could be exploited as a promising biomarker in LUAD treatment.

BackgroundThe development of chimeric antigen receptor (CAR)-T cell therapies for solid tumors has attracted considerable attention, yet their clinical efficacy remains limited. Therefore, various efforts have been made to improve the efficacy of CAR-T cell therapy. As one promising strategy, incorporating the T-cell receptor (TCR) machinery into CAR structures has been reported to improve the efficacy of CAR-T cells in studies using conventional CARs targeting such as EGFR. However, in the case of peptide/major histocompatibility complex (pMHC)-targeted CARs, the advantages of exploiting TCR machinery have not been fully elucidated. We recently developed MAGE-A4-derived pMHC (MAGE-A4 pMHC)-targeted CAR-T cells (MA-CAR-T cells) using a highly specific human scFv antibody against MAGE-A4p230-239/HLA-A*02:01. We aimed to determine whether MAGE-A4 pMHC-targeted CAR-T cells using the TCR machinery (Hybrid MA-TCR-T cells) exhibit superior functionality without compromising antigen specificity.MethodsWe constructed a retroviral vector expressing Hybrid MA-TCR where MAGE-A4 pMHC-specific scFv are fused to human TCR constant chains.ResultsHybrid MA-TCR-T cells demonstrated superior in vitro functions compared with MA-CAR-T cells, while maintaining strict antigen specificity. In addition, functional superiority of Hybrid MA-TCR-T cells to MA-CAR-T cells became more pronounced on repetitive antigen stimulation. In particular, Hybrid MA-TCR-T cells significantly inhibited tumor growth in an immunodeficient mouse model more effectively than MA-CAR-T cells. Ex vivo analyses indicated that their enhanced therapeutic efficacy might result from higher infiltration of functionally active, less differentiated Hybrid MA-TCR-T cells in tumor tissues.ConclusionsThese findings suggest that leveraging the TCR machinery is a promising strategy for enhancing pMHC-targeted CAR-T cell therapy for solid tumors, potentially leading to more effective treatments.

Chimeric antigen receptor engineered T cell (CAR-T) therapy has high therapeutic efficacy against blood cancers, but it has not shown satisfactory results in solid tumors. Therefore, we examined the therapeutic effect of CAR-T therapy targeting carcinoembryonic antigen (CEA) in pancreatic adenocarcinoma (PDAC). CEA expression levels on the cell membranes of various PDAC cell lines were evaluated using flow cytometry and the cells were divided into high, medium, and low expression groups. The relationship between CEA expression level and the antitumor effect of anti-CEA-CAR-T was evaluated using a functional assay for various PDAC cell lines; a significant correlation was observed between CEA expression level and the antitumor effect. We created orthotopic PDAC xenograft mouse models and injected with anti-CEA-CAR-T; only the cell line with high CEA expression exhibited a significant therapeutic effect. Thus, the therapeutic effect of CAR-T therapy was related to the target antigen expression level, and the further retrospective analysis of pathological findings from PDAC patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. Therefore, CEA expression levels in biopsies or surgical specimens can be clinically used as biomarkers to select PDAC patients for anti-CAR-T therapy.

The benefits of CAR-T therapy could be expanded to the treatment of solid tumors through the use of derived autologous αβ T cell, but clinical trials of CAR-T therapy for patients with solid tumors have so far been disappointing. CAR-T therapy also faces hurdles due to the time and cost intensive preparation of CAR-T cell products derived from patients as such CAR-T cells are often poor in quality and low in quantity. These inadequacies may be mitigated through the use of third-party donor derived CAR-T cell products which have a potent anti-tumor function but a constrained GVHD property. Vγ9Vδ2 TCR have been shown to exhibit potent antitumor activity but not alloreactivity. Therefore, in this study, CAR-T cells were prepared from Vγ9Vδ2 T (CAR-γδ T) cells which were expanded by using a novel prodrug PTA. CAR-γδ T cells suppressed tumor growth in an antigen specific manner but only during a limited time window. Provision of GITR co-stimulation enhanced anti-tumor function of CAR-γδ T cells. Our present results indicate that, while further optimization of CAR-γδ T cells is necessary, the present results demonstrate that Vγ9Vδ2 T cells are potential source of 'off-the-shelf' CAR-T cell products for successful allogeneic adoptive immunotherapy.

Supplementary Figure 1 from Imatinib Mesylate Induces Quiescence in Gastrointestinal Stromal Tumor Cells through the CDH1-SKP2-p27Kip1 Signaling Axis

Supplementary Data from Two Distinct Mechanisms of Augmented Antitumor Activity by Modulation of Immunostimulatory/Inhibitory Signals

Abstract Background The impressive success of chimeric antigen receptor (CAR)-T therapy in the treatment of hematologic tumors is leading researchers/clinicians to broaden the possible application of this approach to patients with solid tumors, especially patients with pancreatic ductal adenocarcinoma (PDAC). However, CAR-T cell therapy faces disappointing clinical results in solid tumors due to hurdles unique to solid, but not hematologic, tumors, which include restricted trafficking and limited infiltration into tumors, non-durable persistence, and T cell exhaustion. Since the tumor microenvironment composition varies between tumor type meaning CAR-T cells again face a unique set of challenges in each solid tumor, the major hurdle imposed by PDAC on CAR-T cell therapy remains elusive.Methods In this study, we used immunodeficient NOD/Shi-scid, IL-2RγKO Jic mice orthotopically transplanted with human pancreatic tumor cell line, BxPC-3, and transferred with CAR-T cells specific to carcinoembryonic antigen (CEA), a model that allows analysis of quantitative, spatial and functional dynamics of CAR-T cells in tumor tissues unique to PDAC.Results CAR-T cells suppressed tumor growth of orthotopic human PDAC tumors, but only within a limited time window. Contrary to previous studies that demonstrated a limited persistency and infiltration of CAR-T cells in many solid tumors, they persist and accumulated in PDAC tumor tissues, but CAR-T cells become progressively lost ability to control tumor growth. Ex vivo analysis revealed that CAR-T cells that had been recovered at different time points from mice bearing an orthotopic PDAC tumor exhibited a gradual loss of tumor reactivity. This loss of tumor reactivity of CAR-T cells was associated with the increased expression of AMP-activated protein kinase and Mitofusin1.Conclusions The present results demonstrate the potential effectiveness of CAR-T cells in targeting CEA for the treatment of PDAC. However, optimization of CAR-T cells, especially in the areas of resistance to exhaustion is required.